Bottom Line:
Certain intragenic highly conserved elements have been associated with regulating levels of core components of the spliceosome and alternative splicing of downstream genes.These mutations cause increased inclusion of the alternative exon and decreased overall expression of SNRPB.We provide evidence for the functional importance of this conserved intragenic element in the regulation of alternative splicing and development, and suggest that the evolution of such a regulatory mechanism has contributed to the complexity of mammalian development.

ABSTRACTElucidating the function of highly conserved regulatory sequences is a significant challenge in genomics today. Certain intragenic highly conserved elements have been associated with regulating levels of core components of the spliceosome and alternative splicing of downstream genes. Here we identify mutations in one such element, a regulatory alternative exon of SNRPB as the cause of cerebro-costo-mandibular syndrome. This exon contains a premature termination codon that triggers nonsense-mediated mRNA decay when included in the transcript. These mutations cause increased inclusion of the alternative exon and decreased overall expression of SNRPB. We provide evidence for the functional importance of this conserved intragenic element in the regulation of alternative splicing and development, and suggest that the evolution of such a regulatory mechanism has contributed to the complexity of mammalian development.

f2: SNRPB mutations in the alternative (alt) PTC-containing exon cause increased exon inclusion.(a,b) Cloning of the alternative exon with either the chr20:g.2447951C>G (mut1) or chr20:g.2447847G>T (mut2) mutation into a splicing minigene reporter and transfection into HEK297 cells shows 78% and 80% exon inclusion, respectively, compared with 23% for the wild-type (WT) sequence. Results shown are from one representative experimental replicate of three. A 100-bp DNA ladder was used as a size marker in a. NT, no template control. (c) Patient fibroblasts with the chr20:g.2447951C>G (pt 1), chr20:g.2447952C>G (pt 2) and chr20:g.2447847G>T (pt 3) mutations show increased expression of the PTC-containing transcript by qRT–PCR. (d) The same three patients show decreased total SNRPB expression by qRT–PCR. In c,d, the grey columns represent the normalized average expression from three anonymous controls (Ctrl). The experiment was performed three times. For b–d, statistical significance was determined with a Student’s t-test. Bars indicate s.d. * indicates 0.005<P<0.05, ** indicates 0.0005<P<0.005 with *** indicating P<0.001 and ****P<0.0001.

Mentions:
SNRPB encodes the protein isoforms SmB and SmB′, which are core components of the U1, U2, U4/U6 and U5 small ribonuclear protein (snRNP)20 subunits of the major spliceosome. The highly conserved alternative exon within the second of six introns in SNRPB contains a PTC and has been shown to auto-regulate SNRPB levels through NMD18. The alternate exon, which has a sub-optimal 5′ splice site, is less frequently included when U1 snRNP levels are low as a result of SmB/B′ depletion18. Conversely, it is more frequently included with SmB/B′ overexpression16. We hypothesized that the mutations identified within this exon would alter the homeostatic balance between the coding full-length mRNA and alternative exon-containing transcripts targeted for degradation. Thus, we determined the effect of two of the alternative exon mutations using a splicing reporter minigene assay21. In the presence of the wild-type exon, 23% of all transcripts include this alternative exon, while introduction of either the chr20:g.2447951C>G or chr20:g.2447847G>T mutation shifts the proportion to 78% and 80%, respectively (Fig. 2a,b). Inclusion of the alternative PTC-containing exon was also assessed by quantitative reverse transcription PCR (qRT–PCR) in patient fibroblasts with the chr20:g.2447951C>G, chr20:g.2449752C>G, and chr20:g.2447847G>T mutations. Expression of the PTC-containing transcript increased, whereas overall expression of SNRPB decreased compared with control cells (Fig. 2c,d).

f2: SNRPB mutations in the alternative (alt) PTC-containing exon cause increased exon inclusion.(a,b) Cloning of the alternative exon with either the chr20:g.2447951C>G (mut1) or chr20:g.2447847G>T (mut2) mutation into a splicing minigene reporter and transfection into HEK297 cells shows 78% and 80% exon inclusion, respectively, compared with 23% for the wild-type (WT) sequence. Results shown are from one representative experimental replicate of three. A 100-bp DNA ladder was used as a size marker in a. NT, no template control. (c) Patient fibroblasts with the chr20:g.2447951C>G (pt 1), chr20:g.2447952C>G (pt 2) and chr20:g.2447847G>T (pt 3) mutations show increased expression of the PTC-containing transcript by qRT–PCR. (d) The same three patients show decreased total SNRPB expression by qRT–PCR. In c,d, the grey columns represent the normalized average expression from three anonymous controls (Ctrl). The experiment was performed three times. For b–d, statistical significance was determined with a Student’s t-test. Bars indicate s.d. * indicates 0.005<P<0.05, ** indicates 0.0005<P<0.005 with *** indicating P<0.001 and ****P<0.0001.

Mentions:
SNRPB encodes the protein isoforms SmB and SmB′, which are core components of the U1, U2, U4/U6 and U5 small ribonuclear protein (snRNP)20 subunits of the major spliceosome. The highly conserved alternative exon within the second of six introns in SNRPB contains a PTC and has been shown to auto-regulate SNRPB levels through NMD18. The alternate exon, which has a sub-optimal 5′ splice site, is less frequently included when U1 snRNP levels are low as a result of SmB/B′ depletion18. Conversely, it is more frequently included with SmB/B′ overexpression16. We hypothesized that the mutations identified within this exon would alter the homeostatic balance between the coding full-length mRNA and alternative exon-containing transcripts targeted for degradation. Thus, we determined the effect of two of the alternative exon mutations using a splicing reporter minigene assay21. In the presence of the wild-type exon, 23% of all transcripts include this alternative exon, while introduction of either the chr20:g.2447951C>G or chr20:g.2447847G>T mutation shifts the proportion to 78% and 80%, respectively (Fig. 2a,b). Inclusion of the alternative PTC-containing exon was also assessed by quantitative reverse transcription PCR (qRT–PCR) in patient fibroblasts with the chr20:g.2447951C>G, chr20:g.2449752C>G, and chr20:g.2447847G>T mutations. Expression of the PTC-containing transcript increased, whereas overall expression of SNRPB decreased compared with control cells (Fig. 2c,d).

Bottom Line:
Certain intragenic highly conserved elements have been associated with regulating levels of core components of the spliceosome and alternative splicing of downstream genes.These mutations cause increased inclusion of the alternative exon and decreased overall expression of SNRPB.We provide evidence for the functional importance of this conserved intragenic element in the regulation of alternative splicing and development, and suggest that the evolution of such a regulatory mechanism has contributed to the complexity of mammalian development.

ABSTRACTElucidating the function of highly conserved regulatory sequences is a significant challenge in genomics today. Certain intragenic highly conserved elements have been associated with regulating levels of core components of the spliceosome and alternative splicing of downstream genes. Here we identify mutations in one such element, a regulatory alternative exon of SNRPB as the cause of cerebro-costo-mandibular syndrome. This exon contains a premature termination codon that triggers nonsense-mediated mRNA decay when included in the transcript. These mutations cause increased inclusion of the alternative exon and decreased overall expression of SNRPB. We provide evidence for the functional importance of this conserved intragenic element in the regulation of alternative splicing and development, and suggest that the evolution of such a regulatory mechanism has contributed to the complexity of mammalian development.